Supplemental Digital Content is available in the text 11C-Pittsburgh compound B positron emission tomography (11C-PIB-PET) may play a role in stratifying patients with MCI into risk levels for developing AD. However, the predictive accuracy of amyloid imaging for the progression from MCI to AD with different follow-up durations has not yet been systematically evaluated. A formal systematic evaluation of the sensitivity, specificity, and other properties of 11C-PIB-PET was performed.In the past decade, amyloid deposition has been shown to begin many years before the clinical symptoms of dementia in mild cognitive impairment (MCI) due to Alzheimer disease (AD). Longitudinal studies with different follow-up durations have suggested that 11C-PIB-PET for predicting conversion to AD in patients with MCI and to determine whether long-term follow-up has a positive effect on predictive accuracy.This study aimed to systematically review and meta-analyze published data on the diagnostic performance of 11C-PIB-PET in individual studies were calculated, and the studies underwent meta-analysis with a random-effects model. A summary receiver-operating characteristic curve (SROC) was constructed with the Moses-Shapiro-Littenberg method. Pooled estimates of sensitivity, specificity, positive likelihood ratio (LR+), negative likelihood ratio (LR−), diagnostic odds ratio (DOR), and the SROC curve of each subgroup were determined. Heterogeneity was tested, and potential sources for heterogeneity were explored by assessing whether certain covariates significantly influenced the relative DOR.Relevant studies were systematically identified through electronic searches, which were performed in MEDLINE (OvidSP), EMBASE (OvidSP), BIOSIS Previews (ISI Web of Knowledge), Science Citation Index (ISI Web of Knowledge), PsycINFO (Ovid SP), and LILACS (Bireme). The methodological quality of each study was assessed by QUADAS-2. Sensitivities and specificities of 11C-PIB-PET for predicting conversion to AD ranged from 83.3% to 100% and 41.1% to 100%, respectively, with pooled estimates of 94.7%  and 57.2% (95% CI: 50.1%–64.2%), respectively. Moderate heterogeneity was observed for specificity between the included studies (I2 = 42.1%). The pooled estimates for the long-term follow-up subgroup were 95.5% sensitivity (95% CI: 84.5%–99.4%) and 72.4% specificity (95% CI: 59.1%–83.3.8%), whereas the pooled estimates for the short-term follow-up subgroup were 94.4% sensitivity (95% CI: 88.2%–97.9%) and 51.0% specificity (95% CI: 42.6%–59.5%). Homogeneity in each subgroup was significantly higher than that of the included studies, and most diagnostic indicators in the long-term follow-up subgroup were far superior to those in the short-term follow-up subgroup or the entire group.Eleven eligible studies consisting of a total of 352 patients with MCI at baseline were included. Overall, the studies were of moderate-to-high methodological quality. The sensitivity and specificity of Not all of the methodological quality scores of studies included in this systematic review were high.11C-PIB-PET for predicting the progression from MCI to AD. In particular, the specificity, which reflects the exploratory nature of the use of amyloid imaging to identify the process of MCI to AD, was improved with a longer follow-up period.Current evidence suggests that prolongation of the follow-up duration tended to yield greater accuracy of  The pathology of AD is present in the majority of cases of dementia. As the dominant or sole pathology, it accounts for over 50% of dementia. In some countries, the burden of AD dementia appears to be increasing faster than is generally assumed by the international health community.2 However, the strength of the correlation between cognitive impairment and the pathological features of AD varies both with age and with each pathological feature. A significant number of individuals without clinical evidence of AD exhibit imaging evidence of amyloid deposition.3An increased cerebral β-amyloid (Aβ) burden in the form of fibrillar plaques or soluble oligomers and neurofibrillary tangles (NFTs) in the brain has been suggested to be the primary cause of brain degenerative changes and progressive cognitive deterioration in AD.5 in which the patient has a degree of cognitive impairment greater than expected for their age but is not impaired in function. MCI is a heterogeneous condition that has been defined using clinical criteria by Petersen (1999).5 More than a dozen different definitions have been used to describe cognitive impairment that exceeds normal impairment and is qualitatively different from normal aging. While remaining attentive to the widely differing prognostic implications of the differing terms, in this review, the term MCI will be used to collectively describe the 16 conditions included in Matthews et al's study (2008).7 Four outcomes are observed for those within an MCI population: progression to AD dementia, progression to another dementia, maintaining stable MCI, or recovery. The National Institute on Aging (NIA) and the Alzheimer Association (AA) have published research criteria for MCI due to AD that incorporate the use of biomarkers to assess the likelihood that the MCI syndrome is due to the underlying pathophysiology of AD.8Before patients with AD exhibit typical clinical symptoms of dementia, they present with a stage known as mild cognitive impairment (MCI),9 Although there is no generally accepted diagnostic criterion sufficiently specific for predicting who will rapidly convert to AD among patients with MCI prior to the onset of symptoms in clinical practice, it is notable that amyloid deposition starts many years before the clinical symptoms of dementia in MCI due to AD are evident.10 Molecular neuroimaging, such as amyloid imaging, is useful not only to diagnose different subtypes of dementia but also for the early detection of AD and other neurodegenerative diseases.12 Amyloid positron emission tomography (PET) tracers provide a quantitative in vivo measure of the insoluble cortical Aβ load. To date, the most studied and validated PET marker of Aβ aggregation is 11C-Pittsburgh compound B (11C-PIB), which shows a nanomolar affinity for the extracellular and intravascular fibrillar deposits of Aβ and a low affinity toward the amorphous amyloid deposits, soluble Aβ, and intracellular NFTs.1311C-PIB-PET imaging may be essential for testing an Aβ immunotherapy drug on MCI patients with evidence of brain Aβ pathology, and it appears to be useful in assessing the effects of pre-dementia phase preventive treatments for potential AD on cortical fibrillar Aβ load in vivo. Therefore, proper application of 11C-PIB-PET would be useful to predict the conversion of MCI, which may have many prospects in clinical practice.15The disappointing clinical trial results regarding modifying therapies in patients with AD suggest that the treatment strategy should be targeted to the MCI stage.11C-PIB-PET may play a role in stratifying patients with MCI into their risk of developing AD and that as longitudinal studies continue to accumulate data, 11C-PIB-PET may become useful for predicting future clinical conditions, such as the risk of transitioning to AD. However, these studies require further replication and analysis in a pooled meta-analysis.17 Given the variability in their results and methodological quality, it is challenging to draw definitive and confident conclusions from these longitudinal studies with different follow-up durations. Additionally, recent research has suggested that the follow-up period may play an important role in the diagnostic accuracy for progressing from MCI to AD.18 Therefore, the aims of this meta-analysis were to systematically review the diagnostic efficiency of amyloid PET imaging using 11C-PIB-PET to predict the conversion to AD in patients with MCI; to explore whether different lengths of follow-up (short-term and long-term) have an effect on the prognostic value in longitudinal studies; and to discuss the relationship between the length of follow-up and predictive accuracy.Some longitudinal studies with different follow-up durations have suggested that 11C Pittsburgh or PiB-PET or 11C PiB-PET OR amyloid ligand or [11C]PiB) AND (positron emission tomography or PET) AND (amyloid imaging or beta-amyloid or amyloid or amyloid-β or amyloid deposition) AND (mild cognitive impairment or MCI) AND (Alzheimer’ disease or Alzheimer disease dementia). The search was limited to articles including human subjects. Reference lists of retrieved articles and any relevant systematic reviews were screened for additional studies. No language restriction was applied to the electronic searches.Relevant studies were identified through electronic searches, which were performed in MEDLINE (OvidSP), EMBASE (OvidSP), BIOSIS Previews (ISI Web of Knowledge), Science Citation Index (ISI Web of Knowledge), PsycINFO (Ovid SP), and LILACS (Bireme), for eligible studies published from 1999 (criteria for the diagnosis of MCI was proposed by Petersen [1999)]) through February 2014. The search strategy was performed with a combination of terms for titles and abstracts:  result of MCI was identified at follow-up, and the study included at least 10 MCI participants.21; furthermore, core clinical criteria for MCI, proposed by the NIA-AA workgroup, were also acceptable.8 These criteria include subjective complaints, a decline in memory objectively verified by neuropsychological testing in combination with a history from the patient, a decline in other cognitive domains, no or minimal impairment of activities of daily living, and not meeting the criteria for dementia. Therefore, the eligible participants underwent a number of tests, for example, neuropsychological tests for cognitive deficits and checklists for activities of daily living, prior to study entry. MCI participants did not use acetylcholinesterase inhibitors or other neuropsychopathic medications during follow-up.MCI participants at baseline met the Petersen criteria or revised Petersen criteriaThe diagnoses of AD met probable National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria of AD or the Diagnostic and Statistical manual of Mental disorders (DSM) and International Classification of Diseases (ICD)'s definitions for AD.11C-PIB positivity, and those with 11C-PIB negativity using validated PET imaging at baseline. The definition of 11C-PIB positivity was that 11C-PIB ligand uptake exceeded a certain threshold in the PET imaging protocol and vice versa.MCI participants could be grouped into those with Follow-up periods for all MCI participants were at least 12 months, and clinical outcomes of MCI participants could be obtained at the last follow-up.Studies were included if they fulfilled the following criteria:Two authors (ZDM and ZS) performed the first assessment of the search results to remove the obvious non-relevant studies. Two authors (MY and FJ) independently reviewed the remaining abstracts and titles identified by the database searches for potentially eligible studies. If one or both reviewers considered the study potentially eligible, the full manuscript was evaluated against the inclusion criteria by both reviewers. Discrepancies were resolved by consensus. When necessary, a third arbitrator resolved disagreements not able to be resolved through discussion.Basic clinical and demographic details of participants at baseline including age, sex, education, Mini-Mental State Examination (MMSE) score, apolipoprotein E(APOE)-ε 4 carrier status, number of subjects, MCI clinical criteria, MCI subtypes, sources of referral, participant recruitment, and sampling procedures. If there were several baseline time points, only data from the initial time point were used.11C-PIB-PET imaging consisting of the 11C-PIB test administration method, including the time between 11C-PIB injection and PET acquisition, thresholds used to define positive and negative tests, 11C-PIB dose, measures of 11C-PIB amyloid retention, and image analysis of discriminating brain regions.Protocols for 11C-PIB-PET imaging was performed to defining AD dementia by reference standard , prevalence or proportion of the population developing AD, and cumulative and per-year conversion rate with severity, if described.Procedures for the conversion to AD, including the reference standard of AD dementia used in included studies, the duration of follow-up from the time that The following information was extracted from each eligible study:11C-PIB-positive image results determined by PET, and the subsequent conversion to AD was confirmed at follow-up. TN was defined as the number of MCI patients who had 11C-PIB-negative image results determined by PET, and MCI due to AD was not confirmed by the end of follow-up. FP was defined as the number of MCI patients with 11C-PIB-positive image results that did not progress to AD during the follow-up. FN was defined as the number of MCI patients with negative imaging results, but the conversion to AD was eventually confirmed.The data were extracted independently by 2 blinded review authors (ZS and MY) and included the number of patients with MCI that converted to AD, number of nonconverters, number of true-positives (TPs), false-positives (FPs), false-negatives (FN), true-negatives (TN), sensitivity, specificity, positive likelihood ratio (LR+) and negative likelihood ratio (LR−). TP was defined as the number of MCI patients who had 11C-PIB-positive diagnosis determined by PET (TP/[TP + FN]). Specificity was defined as the proportion of MCI nonconverters who received the correct 11C-PIB-negative diagnosis determined by PET (TN/[TN + FP]). LR+ was calculated as sensitivity/(1-specificity), and LR− was calculated as (1-sensitivity)/specificity. LR+ indicates how much to increase the probability of progression to AD if the 11C-PIB-PET test of an MCI patient is positive, and LR− indicated how much to decrease the probability of progression to AD if the 11C-PIB-PET test of an MCI patient is positive. If the above data were unavailable from original article, the data were calculated by extracting the relevant data.The sensitivity and specificity of individual studies were extracted and calculated using 2 × 2 contingency tables. Sensitivity was defined as the proportion of MCI converters who received the correct When a study did not present all relevant data necessary for creating a 2 × 2 table, we contacted the authors directly to request further information. The numbers lost to follow-up were recorded for each included study. We also extracted the data necessary for assessing quality, as defined below.11C-PIB-PET to predict MCI to AD conversion.The time interval over which progression from MCI to AD occurs is very important; therefore, all included studies were divided into several subgroups according to different follow-up periods, if necessary (please see Thresholds Effect and Investigations of Heterogeneity). Specifically, we chose 1 year as the minimum period of delay in verifying the diagnosis . The analysis was segmented into separate follow-up mean periods for the delay in verification: 1 year to <2 years; 2 to <4 years; and >4 years. This segmentation was performed to explicitly compare short- and long-term follow-up intervals and determine whether a long-term follow-up interval is more beneficial for using 22 as recommended by the Cochrane Collaboration. QUADAS-2 evaluates the risk of bias and concerns regarding the applicability for patient selection, index tests, reference standards, and risk of bias in the domain of study flow/timing. All included articles were analyzed in terms of patients (newly diagnosed MCI), index test (11C-PIB-PET), reference standard (AD dementia), and flow and timing. Studies were scored as “L” for low risk of bias/low concerns regarding applicability, “H” for high risk/high concerns, and “U” for unclear items for each domain. All domains with at least 1 negative response were scored as H regarding applicability, whereas domains with no negative responses but at least 1 unsure response were scored as U. Domains with no negative and no unsure responses were scored as L. Two blinded independent raters (GY and LJ) performed the QUADAS-2 assessment. Any disagreements were resolved by consensus or arbitration. For each individual study, the final results of the quality assessment were tabulated.The methodological quality of the included studies was assessed using the Revised Quality Assessment of Studies of Diagnostic Accuracy Included in Systematic Reviews (QUADAS)-2 tool,11C-PIB-PET at baseline and being cognitively progressive or stable during the follow-up interval. To calculate the log OR , we added 0.5 to each cell in any 2 × 2 table with a value of zero.For each study, we constructed a 2 × 2 contingency table consisting of TP, FP, FN, and TN results, in which all participants were classified as presenting with positive or negative If no threshold effect was observed, the pooled indices of sensitivity and specificity with the corresponding 95% confidence intervals (CIs) were calculated using weighted averages according to the sample size of each study. The pooled estimates of LR+, LR−, and diagnostic odds ratio (DOR) were computed using the DerSimonian and Laird method based on a random-effects model.∗ on a symmetric ROC curve, which is a global measure of test accuracy.Subsequently, exploratory analyses were conducted by plotting estimates of sensitivity and specificity from each study on forest plots, and the results of the individual studies were also displayed in receiver-operating characteristic (ROC) space. A weighted symmetric summary ROC curve (SROC) with a 95% CI was computed with the Moses-Shapiro-Littenberg method according to different situations, and the quantitative value of the area under the curve (AUC) ± standard error (SE) was also calculated. Furthermore, we also defined the maximum joint sensitivity and specificity as point Q11C-PIB-PET imaging test accuracy was evaluated according to the target condition. Currently, there is a set of acknowledged thresholds for defining 11C-PIB-PET positivity for MCI due to AD and AD dementia, and therefore, the estimates of diagnostic accuracy reported in primary studies were likely to be based on data-driven threshold selection23 unless prespecified. For studies in which researchers did not report a criterion for positive 11C-PIB-PET imaging results, the optimum threshold value was used as the reference standard, which was calculated by constructing a ROC curve from the study.24 Heterogeneity between the results of individual studies was tested by the inconsistency index (I2), which describes the percentage of total variance due to heterogeneity rather than chance across different studies. A zero percentage index indicates no heterogeneity, whereas 25%, 50%, and 75% indicate low, moderate, and high heterogeneity, respectively. To determine whether a potential source of heterogeneity resulted from certain covariates , meta-regression analysis was performed on the condition sufficient studies with available data were available. We considered variates explanatory if their regression coefficients were statistically significant (P < 0.05). In addition, if any level of heterogeneity or a significant covariate was identified, further subgroup analyses were performed.To determine whether researchers may have used different thresholds to define positive and negative test results (either explicitly or implicitly), a Spearman correlation coefficient between the logit of sensitivity and logit of 1-specificity was calculated to assess the presence of a threshold effect. A strong positive correlation (Spearman ρ > 0.6) suggested the presence of a threshold effect.To evaluate the robustness of the meta-analysis, a sensitivity analysis of each subgroup was performed by excluding studies individually starting from the minimum to the maximum follow-up period to assess the influence of an individual study on the DOR. We also investigated the effect of prespecification of threshold and amyloid positivity on diagnostic accuracy by performing sensitivity analyses. Consistent results indicated stronger evidence of an effect and of study generalizability.25 The above opinions were supported by Cochrane systematic reviews of diagnostic test accuracy.29Publication bias was not investigated due to current uncertainty regarding how performance bias operates in test accuracy studies and the interpretation of existing analytical tools, such as funnel plots.All statistical analyses were executed with Stata, version 11  and Meta-DiSc statistical software, version 1.4 .11C-PIB but other radiotracers  that could not be differentiated (n = 3); no threshold was used (changes in 11C-PIB levels measured over time) (n = 10) .The computer-aided search revealed 2621 articles from electronic databases (APPENDIX 1). After de-duplication and first-assessments of abstracts, 89 articles remained and were screened for eligibility for this meta-analysis. After the first review of the full-text article, 55 articles remained. In a second round of screening of full-text articles, 44 articles were excluded for following reasons: no extractable data for constructing 2 × 2 tables to calculate sensitivity and specificity (n = 4); participants exhibited other conditions in addition to MCI, which could not be differentiated at baseline (n = 7); reference standards were for not only AD dementia but also other types of dementia, such as frontotemporal dementia, dementia with Lewy bodies, and dementia that could not be differentiated (n = 4); no delayed verification study (n = 8); results from a combination of other diagnostic modalities, such as neuropsychological tests, that could not be differentiated (n = 4); multiple publications, that is, the same data overlapped with another eligible study consisting of a larger number of patients in the study (n = 4); the tracers used in PET imaging were not only 39 Of the 352 participants with analyzable data, 151 developed AD, and 9 developed non-AD-type dementia. The remaining 26 participants were reported to be lost to follow-up. The majority (n = 18) of participants were missing from a single study.36Thus, 11 studies remained, with a total sample size of 378 participants with MCI at baseline.39 the participants with MCI fell into 2 subgroups according to several criteria, such as sources of subjects, follow-up findings (progressive or stable), and PET imaging assessments (positive or negative). To achieve consistent expression, some quantitative variables at baseline from both subgroups were expressed as the mean ± standard deviation (M ± SD). The weighted pooled M ± SD was calculated using statistical methods. Two methods were utilized to measure the amyloid retention in Jack et al’ study (2010)33: transforming cerebrospinal fluid (CSF) Aβ42 measures into calculated 11C-PIB measures and 11C-PIB-PET imaging. The study pooled measures of Aβ from either source, and the number of amyloid-positive subjects that progressed from MCI to AD in whom amyloid retention was measured by 11C-PIB-PET was not reported in that article. Although transforming CSF Aβ42 into calculated 11C-PIB was shown to be an effective alternative to measuring amyloid load,40 to avoid the potential risk of bias, the authors were contacted, and the raw data from subjects with MCI measured by 11C-PIB-PET imaging were available. In addition, although some usable data were also obtained for 2 studies37 by contacting the authors of the studies, screening the reference lists of these articles did not result in other potentially relevant articles. The study sizes were small and ranged from 10 to 68. Five studies reported a mean age of >70 years,39 and 5 articles reported a mean age <70 years.37 Only 1 study32 reported a range of ages.In some studies,34 also used DSM-IV criteria. One study36 did not explicitly state what reference standard was used at follow-up. The duration of follow-up was reported as the mean and SD, median, minimum duration, maximum duration, or range. The length of follow-up varied substantially but was mostly within a range of 18 to 50 months. The longest follow-up period of participants with MCI was found in Kemppainen et al’ study (2014).18 Because a significant difference was observed in the durations of follow-up between the long- and short-term follow-up studies, these included studies were divided into 2 subgroups classified as long-term and short-term according to whether the length of follow-up was >2 years. Six studies39 were classified as short-term, and 537 were classified as long-term. The demographic and patient characteristics of the included studies are summarized in Table 39All of the studies were published within the last 5 years 2009–2014). Most studies (7/11) were conducted in Europe: 2 were conducted in Asia and the Pacific, and 2 were conducted in the United States. Nine included studies applied the NINCDS-ADRDA criteria as a reference standard for AD dementia; Koivunen et al4. Most s11C-PIB-PET scans were performed and interpreted. Table 3 and Table 4  summarize the data regarding the 11C-PIB-PET protocols.The included studies varied markedly in how the http://links.lww.com/MD/A79). The overall methodological quality of the studies according to the QUADAS-2 scores is displayed in Table 6 .The review authors’ judgments regarding each methodological quality item for each included study are presented in Table 5 . The SROC curve showed a Q value of 0.81 ± 0.03 (±SE) and an AUC of 0.88 ± 0.03 (±SE) , which suggested that there was no threshold effect. Meta-regression to explore potential sources of heterogeneity was not performed because the available data for most covariates were insufficient for conducting this analysis.The inconsistency index values were 0%, 42.1%, 22.9%, 0%, and 0% for sensitivity, specificity LR+, LR−, and DOR, respectively, revealing a possible low-to-moderate heterogeneity between the included studies. The cumulative conversion rate was 42.9% (151/352). For the included studies, the sensitivities were between 83.3% and 100%, the specificities were between 42.1% and 100%, the pooled sensitivity was 94.7% (95% CI: 89.8%–97.7%), the pooled specificity was 57.2% (95% CI: 50.1%–64.2%), the pooled LR+ was 2.08 (95% CI: 1.71–2.55), and the pooled LR− was 0.15 (95% CI: 0.08–0.26) , 51.0% specificity (95% CI: 42.6%–59.5%) , 1.83 LR+ (95% CI: 1.54–2.18) and 0.16 LR− (95% CI: 0.08–0.34). The pooled diagnostic accuracy was 69.6%, and the random-effects model estimated an overall DOR of 13.01 (95% CI: 5.48–30.87) (P = 0.48). The SROC curve showed a Q value of 0.81 ± 0.03 (±SE) and an AUC of 0.88 ± 0.03 (±SE) , indicating that there was no threshold effect.The data extracted from this subgroup had significant homogeneity, with inconsistency index values of 0%, 1.7%, 0%, 0%, and 0% for sensitivity, specificity, LR+, LR−, and DOR, respectively. The cumulative conversion rate was 42.8% (107/250). The sensitivities and specificities of I2 = 0%). The cumulative conversion rate was 43.1% (44/102) in the long-term follow-up subgroup. The overall range of reported sensitivity and specificity was from 88.9% to 100% and 57.9% to 100%, respectively. The pooled estimates for the 11C-PIB-PET data were 95.5% sensitivity (95% CI: 84.5%–99.4%), 72.4% specificity (95% CI: 59.1%–83.3.8%), 2.90 LR+ (95% CI: 1.97–4.27), and 0.12 LR− (95% CI: 0.05–0.32). The pooled diagnostic accuracy was 82.4%, and the overall DOR was 36.88 (95% CI: 9.65–140.96) (P = 0.981) . The area under the SROC curve (±SE) and its Q∗-point (±SE) were 0.93 ± 0.03 and 0.86 ± 0.04, respectively. The summary ROC curve summarizing the accuracy across the 5 studies is shown in Figure P = 0.17), revealing that there was no threshold effect.The data extracted from this subgroup had almost no heterogeneity, with inconsistency index values of 0%, 5.6%, 0%, and 0% for sensitivity, specificity, LR+ and LR−, respectively. Moreover, the DOR was also homogeneous across individual studies ; although metaregression analysis was not performed due to insufficient data, we had sufficient statistical evidence to speculate that the duration of follow-up may be a major cause of the presence of heterogeneity. Additionally, most diagnostic indicators in the long-term follow-up subgroup were superior to those in the short-term follow-up subgroup and the entire group, especially for specificity, LR+, diagnostic accuracy, and DOR, implying that a longer length of follow-up is instrumental in raising the predictive value and diagnostic accuracy of 11C-PIB-PET in the early detection of MCI due to AD. In addition, no significant differences were observed between the 2 subgroups in the cumulative conversion rate of MCI .Notably, the homogeneity of specificity in each subgroup was significantly higher than that of the included studies (http://links.lww.com/MD/A79), which shows that the DORs were significantly altered after removing some studies from each subgroup, suggesting that the diagnosis accuracy was mostly influenced by these studies. We did not observe any obvious impact on our findings.The sensitivity analyses are summarized in Table 7 , the clinical outcomes of these MCI patients would not be changed by the length of follow-up. Thus far, the longest follow-up study with 2 11C-PIB-PET scans (at approximately 2 and 5 years) was conducted by Kemppainen et al.18 Although this study only included 10 MCI participants, it exhibited the highest specificity (100%) and a higher diagnostic accuracy (90%) of positive 11C-PIB-PET scans for predicting MCI to AD conversion at 5 years. Notably, only 1 MCI participant was diagnosed with AD at the time of the scan at 2 years, whereas the others converted between the scan at 2 years and the scan at 5 years. These results differ from the previously reported 42% to 77% specificity of 11C-PIB-PET for predicting short-term MCI to AD conversion.42 The inconsistent estimates may be explained by the longer follow-up period. In addition, recent studies43 have demonstrated that MCI with 11C-PIB negativity at a certain time point did not exclude the possibility of later progression to clinical AD and 11C-PIB positivity.The duration of follow-up may play an important role in predicting conversion to AD. Although the length of follow-up in the included studies was at least 1 year, the variability in the duration of follow-up was considerable, ranging from approximately 1.0 to 5.2 years. In general, given a longer length of follow-up, a higher proportion of MCI patients at baseline will progress to AD, thus affecting the predictive accuracy of 44 Aβ accumulation occurs over 2 to 3 decades and can be detected at low levels with 11C-PIB-PET at 15 to 20 years before the typical levels found in patients with MCI due to AD are observed. Amyloid deposition is a long-term, dynamic process. The relationship between amyloid accumulation and time is considered to have a sigmoid-shaped trajectory, and when the amyloid load reaches a higher level , rates of amyloid accumulation will approach a plateau. MCI is not a direct consequence of amyloid deposition, whereas amyloid deposition is an earlier, upstream pathophysiological event.45 Moreover, MCI due to AD conversion of a given amount of amyloid can be modified by many factors. For example, progression may occur sooner in MCI participants with risk-enhancing characteristics, such as other AD pathologies , low education, or risk amplification genes. Similarly, cognitive decline may be delayed in MCI participants with risk-reducing characteristics, such as high education or protective genes.46 In contrast, these studies suggest that the progression of cognitive status and hippocampal atrophy are not detectable in an individual with MCI due to AD until approximately 5 years before the symptomatic stage of AD.47The above finding is consistent with other studies regarding the relationship between Aβ accumulation and other features of cognitive impairment.48 However, due to the lack of available information, the association between mean conversion rates and the length of follow-up was not well elucidated in this review. Whether the mean conversion rates, which are similar to the rates of amyloid accumulation, have an inverted U shape is unknown.43 According to recent theoretical models,3 longer follow-up durations may yield high cumulative conversion rates in a given timeframe, thus affecting the predictive accuracy of 11C-PIB-PET. Although the relationship between the cumulative conversion rate and length of follow-up was not confirmed in our review, some results obtained from the included studies require further attention. For example, the study18 with the longest length of follow-up (exceeding 5.2 years) had the highest cumulative conversion rate (90.0%); correspondingly, the studies39 with the lowest cumulative conversion rates were in the short-term subgroup. Further studies are needed to confirm this association.Cumulative and mean conversion rates of MCI may be another factor associated with the duration of follow-up, and this factor could have also influenced predictive accuracy. The mean conversion rate of MCI to AD ranged from approximately 8.3% to 29.3% per year (approximately 8.3%–24.9% for the short-term and 17.0%–29.3% for the long-term follow-up subgroup) for the included studies in our meta-analysis, which is similar to the previously reported 8.1% to 28.9% per year.11C-PIB-PET. Amnestic MCI (a-MCI) may be a precursor of AD, whereas nonamnestic MCI (na-MCI) may be a precursor of other types of dementia. Patients with na-MCI are generally believed to have a significantly lower rate of conversion to AD than a-MCI patients. In a longitudinal study with 550 MCI patients,49 MCI with storage memory impairment had the highest and closest risk of conversion to dementia, particularly AD. In contrast, another study found lower conversion rates to AD in patients with a single-domain subtype of MCI compared with those with the multiple-domain type, suggesting that the number of impaired cognitive domains rather than the presence of memory impairment predicted the progression to AD. Furthermore, the MCI subtype was diagnostically unstable.50 In our review, large variations and unavailable data of MCI subtypes were observed in the included studies; Grimmer et al (2013)31 and Wolk et al (2009)39 found no clear association between a particular clinical subtype of MCI and 11C-PIB-PET positivity. However, subjects with multidomain amnestic symptomatology more frequently exhibited a significant clinical decline compared with individuals with isolated memory impairment in Grimmer et al's study (2013),31 which is consistent with a previous study.50 None of the included studies analyzed the underlying correlation between 11C-PIB-PET imaging, conversion rate, and MCI subtypes; therefore, to clarify the correlation between MCI subtypes and brain Aβ burden, further prospective 11C-PIB-PET studies with MCI subtypes should be performed.Some other demographic characteristics of MCI may be underlying factors that modify the conversion rate and predictive accuracy of 39 in both subgroups generated their own optimal thresholds, prespecified or did not prespecify what constituted a positive or negative test, and some reports39 used visual 11C-PIB-PET interpretation. This inconsistency is being addressed currently, although the genesis of uniform imaging analysis techniques and thresholds will not alter the diagnostic accuracy of the test; however, uniform imaging analysis will enable easier integration of results across studies.Notably, some studies11C-PIB-negative patient clinically diagnosed with probable AD.51 In addition, a cohort study52 indicated that plaques and tangles independently contribute to cognitive impairment in AD pathology without any other primary neuropathologic diagnosis. Furthermore, NFT formation must therefore be either unrelated to amyloid plaque formation, be temporally distinct process, or both.53 Some false-negative findings in diagnostic tests may result from these pathological features. Second, some patients with probable MCI by clinical criteria with positive amyloid PET imaging could have had cerebral amyloid angiopathy rather than AD,55 resulting in false-positive results. In addition, those with probable AD may have had multiple brain pathologies, most commonly AD with macroscopic infarcts, followed by AD with neocortical Lewy body disease. Similar to AD, the pathology underlying MCI may also be heterogeneous.56 Currently, it is unknown whether the complicated and mixed pathology influences imaging and diagnosis by 11C-PIB-PET; however, we speculate that the mixed neuropathology of probable AD and MCI may play a role in 11C-PIB-PET-negative MCI converters. Unfortunately, no study categorized their cohort by postmortem pathological examinations in the longitudinal follow-up.Some special pathological types of probable AD can also be used to interpret the false-positive and false-negative results, which are described as follows. First, early AD pathology at autopsy has been reported in an 11C-PIB-PET studies was moderate. An international consensus initiative (the STARDdem Initiative) coordinated by the Cochrane Dementia and Cognitive Improvement Group was recently conducted. This initiative aimed to review the current standard of reporting in diagnostic test accuracy studies and cognitive impairment to generate enhanced guidance in addition to the existing reporting guidelines for diagnostic test accuracy studies.57 This developing research field supports the acceptance of consistent methodologies and reporting, which would assist future reviews of the diagnostic accuracy of tests and their synthesis in meta-analyses. Second, the included studies did not report sufficient information to assess the effects of baseline MCI characteristics  on the predictive value of 11C-PIB-PET for converting to AD. Significant heterogeneity of specificity was observed between the included studies. In the subgroup analysis, we found probable explanations for the heterogeneity, but other factors that possibly contributed to the heterogeneity remain unidentified. Third, the number of included studies was relatively small, and insufficient information regarding follow-up limited more specific segment analyses in this review. Forth, some included studies have a relatively small sample size, and no large-scale prospective validation of 11C-PIB-PET studies with MCI has been conducted, indicating the risk of sampling bias in the included studies.The present meta-analysis had several limitations. First, not all included studies were of high methodological quality. Although the quality of the studies reported in our analysis was generally moderate to high, the overall methodological and reporting quality of all considered 11C-PIB-PET in this emerging literature. We observed that longer duration studies tended to yield greater accuracy and particularly, specificity. Specifically, the observations made here regarding the utility of 11C-PIB-PET in identifying AD prior to the onset of dementia suggest that at the MCI phase, 11C-PIB-PET has great utility. Furthermore, long-term follow-up is beneficial for 11C-PIB-PET to validate predictive accuracy. Future research may indicate that at even earlier stages , Aβ accumulation in the brain will have greater specificity for indicating pathology rather than normal aging. The updated review should determine whether long-term follow-up increases the predictive accuracy of other biomarkers . Moreover, more uniform approaches to thresholds, imaging analysis, study protocols, and particularly, uniformity follow-up duration, may provide a more homogenous estimate than available here.26This is the first such systematic review analyzing the effects of follow-up length on the prognostic value of 18F-fluoro-2-deoxyglucose PET and structural magnetic resonance imaging, 11C-PIB-PET exhibited the best sensitivity but had lower specificity in some predictive accuracy studies with short-term follow-up.58 Our findings provide convincing evidence that long-term follow-up period could compensate for the lower specificity in longitudinal 11C-PIB-PET studies with MCI. Additionally, this observation reflects the predominantly exploratory nature of the use of amyloid imaging to identify disease processes rather than as a diagnostic test with clinical utility. The early detection of MCI due to the AD process is crucial for early therapeutic interventions to delay the onset of the clinical symptoms and slow cognitive decline. By combining our findings with data emerging from other investigators,43 predicting the timeline for an individual with MCI with a given Aβ burden to reach the levels usually noted in AD may be possible. The development of better, more accessible, and more accurate biomarkers with better specificity is urgently required.Compared with


Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein’s topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum’s physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen. Treponema pallidum. Although this obligate human pathogen is a diderm like Gram-negative bacteria, the cell envelope has unique features. Particularly notable are the scarcity of outer-membrane proteins  S. The same value of the protein in the presence of D-glucose was 3.292 [3.288–3.297] S. We can estimate the statistical significance of this difference the F-statistic formalism. If we fix the s-value of the apo-protein to that of the liganded protein and repeat the analysis, we arrive at a χ2 ratio between the two analyses of 1.86. Given the degrees of freedom in this experiment, it is extremely improbable (probability ≈ 10−16) that there is no difference in the s-values.Our initial attempt to examine whether this small difference could be detected was conducted using conventional SV. In experiments carried out simultaneously, we sedimented apo-TpMglB-2 and also the protein in the presence of 100 μM D-glucose. By analyzing the data with a non-interacting discrete species model , we couls normalized by the mean s-value. Thus, when a normalized first moment of the interference signal is plotted as shown in Although the previous SV study strongly suggested a conformational change, we sought a more direct comparison of the two sedimentation rates. Minute sedimentation-coefficient differences are readily detected using a method pioneered in H. Schachman’s lab called “difference sedimentation velocity” . This mes for this same concentration of TpMglB-2, 3.2 S, results in an observed change of approximately 0.06 S. This correlates well with the difference obtained from comparing the s-value of individual experiments and also with the hydrodynamic modeling performed on the structural homolog EcMglB (see above).As shown in An important control experiment in difference SV is to conduct the study with identically prepared solutions in each sector. When applied to TpMglB-2, this approach resulted in a weak negative trend in the first moments, eliciting a Another critical consideration in difference SV experiments is the this see  to be 0.T. pallidum is thought to be microaerophilic[T. pallidum. These facts make the study of the cellular import of D-glucose critical for understanding the lifestyle of the organism. In this report, we found that TpMglB-2, the product of gene tp0684, is very likely the D-glucose-binding protein of an ABC transporter dedicated to the import of this vital molecule. This conclusion identifies this protein as a crucial cog in the machinery of energy acquisition for T. pallidum. TpMglB-2 has an unconventional topology for an LBP  are found mostly in other treponemes or Gram-positive bacteria. A noteworthy commonality among most of these organisms is that their respective LBPs are probably tethered. In the treponemes, these proteins are membrane-tethered in the periplasmic space, and they are probably attached to the outer cell wall in Gram-positive bacteria. These facts raise the intriguing possibility that this divergent topology arose as an adaptation to the necessity of anchoring the proteins to a membrane. No physical or mechanical advantage to this topology is immediately obvious from the structure, but we do note that the N-terminus of EcMglB is near to two sugar-binding amino acids (D14 and F16), whereas the topologically distinct N-terminus of TpMglB-2 is near to only one (N19). Therefore, strain on the N-terminus induced by tethering and Brownian motion could have a more adverse effect on the binding of D-glucose in the T. pallidum is whether TpMglB-2 and its cognate ABC permease/ATPase represent the organism’s only means of glucose uptake. Another protein, the product of tp0545, also has sequence homology to EcMglB and has been termed “TpMglB-1” [tp0545 gene does not appear to be in an operon with other ABC transporter components. Hidden-Markov-model searches [An important question to resolve regarding the biology of pMglB-1” ,37. The E. coli structural homolog of TpMglB-2, EcMglB, is a multifunctional protein. In addition to its role as the LBP for a D-glucose/D-galactose ABC transporter, it is also the chemoreceptor for a well-studied sugar chemotaxis system. We and others [T. pallidum. Indeed, motility and chemotaxis are likely major mechanisms for this spirochete to invade tissues distal from the site of initial infection [As detailed elsewhere , the E. d others ,66,67 hanfection ,69, thustp0684, is clustered with probable transmembrane and ATP-binding elements of the cognate ABC transporter. As no other carbon source has been reliably identified that supports the growth and chemotaxis of T. pallidum, this transporter has implications for the survival and pathogenicity of this bacterium.TpMglB-2 adopts a fold that is common for periplasmic LBPs; however, it displays a hitherto unknown topology for this class of proteins. Careful investigation of the binding and physical characteristics of the protein are consistent with the notion that this protein is acting as the ligand-binding element of an ABC transporter for D-glucose. This assertion is buttressed by noting that the gene for this protein, E. coli, the DNA fragment encoding amino acid residues 11–378  of TP0684 was PCR amplified from T. pallidum genomic DNA by the polymerase incomplete primer extension (PIPE) cloning method using ends-specific primers (PIPE insert). The expression vector, pSpeedET , which encodes an N-terminal TEV-protease cleavable expression and purification hexa-histidine tag (MGSDKIHHHHHHENLYFQG), was PCR amplified with PIPE-vector primers. The PIPE-insert and PIPE-vector was mixed to anneal the amplified DNA fragments together[E. coli HK100 competent cells were transformed with the mixtures (PIPE-vector and insert) and selected for kanamycin resistance on LB agar plates. Cloning junctions/fragments were verified by DNA sequencing. A verified plasmid was then co-transformed with pGroESL  into E. coli BL21 AI (Invitrogen) cells for soluble protein expression. E. coli BL21 AI cells were grown at 37° C in LB medium containing 0.1% (w/v) glucose, 40 μg/mL of kanamycin and 30 μg/mL of chloramphenicol until the cell density reached an A600 of 0.5. The culture was then induced for overnight at 16° C with 0.2% (w/v) L-arabinose. The procedures for expression and purification of the recombinant proteins were essentially as previously described [To produce a non-lipidated, recombinant derivative of TP0684 in  together. E. coliescribed .tp0684 was recloned into a pProEx HTb vector (Invitrogen) and co-transformed with pGroESL (Takara) into a methionine auxotroph E. coli B834 (DE3). The recombinant protein was overproduced and purified as described previously [For the production of selenomethionine labeled protein, eviously .tp0684 sequence using the QuikChange site-directed mutagenesis kit . The mutation was confirmed by DNA sequencing. The mutant protein was expressed and purified as described above. Protein concentrations were determined in buffer A  using spectrophotometry. Extinction coefficients were calculated using the Protparam tool of ExPASy (www.expasy.org).For the construction of the recombinant TpMglB-2 variants, the N19A, W145A, D195A and F313A mutation was individually introduced into the plasmid carrying the wild-type Crystals of TpMglB-2 were obtained by mixing 4 μL of TP0684 (~22 mg/mL in buffer A) with 4 μL of crystallization buffer  PEG MME 5,000) and incubating them over 0.5 mL of the reservoir  for 10 days. The crystals were transferred to the stabilization buffer  PEG MME 5,000, 5% (v/v) ethylene glycol). After about 5 minutes in SB, they were serially transferred to buffers that were the same as SB except that they had higher concentrations of ethylene glycol. The final concentration of ethylene glycol was 25%. After about 1 min in this solution, the crystals were flash-cooled in liquid nitrogen. Toward solving the phase problem, crystals of a selenomethionyl derivative of TpMglB-2 (SeTpMglB-2) were grown by mixing 4 μL of SeTpMglB-2 (~9 mg/mL in buffer A) with 4 μL of crystallization buffer  PEG 10,000) and incubating them over 0.5 mL of reservoir solution containing the crystallization buffer for 7 days. The crystals were transferred to the stabilization buffer 2  PEG 10,000, 5% (v/v) ethylene glycol) and cryoprotected/flash cooled as above.1 and diffracted X-rays with a dmin spacing of 2.05Å. The data were reduced and scaled using HKL3000 [dmin spacing, but exhibited significantly divergent unit-cell constants. Diffraction data from the selenomethionine-derivatized crystals were reduced and scaled using HKL3000. The GUI interface in that program was utilized to perform a substructure solution .All X-ray diffraction data were acquired at beamline 19-ID of the Structural Biology Center at Argonne National Laboratories. Native TpMglB-2 crystals had the symmetry of space group C222 HKL3000 . Phase dg SHELXD ), site rg SHELXD ), densitg SHELXD ; no non-g SHELXD ). This sg SHELXD . The modg SHELXD . After ag SHELXD  using pogeometry , and it TpMglB-2 was dialyzed exhaustively against Assay Buffer . The sugars to be titrated were dissolved in the same buffer. All titrations were carried out in an iTC200 calorimeter . A typical titration scheme featured 19 or 20 2-μL injections of 500 μM of the monosaccharide into 50 μM protein in the ca. 200-μL stirred interaction cell. However, the concentrations were sometimes altered to sample a wider range of concentration space. Three tM) and partial-specific volume (HYDROPRO  was usedSEDNTERP  and inpuSEDNTERP  denuded SEDNTERP  was usedc(s) method in SEDFIT [All AUC experiments were carried out at 20° C in a Beckman-Coulter Optima XL-I centrifuge , and Assay Buffer was used in all cases. Charcoal-filled Epon centerpieces with 1.2-cm path-lengths were housed between two sapphire windows in a standard aluminum housing. After introduction to the centerpiece sectors, all sample cells were placed in an An50-Ti rotor that was subsequently incubated at the experimental temperature under vacuum in the centrifuge for at least 2.5 h prior to the initiation of rotation. The rotor speed was 50,000 rpm for all AUC studies. For normal SV experiments, 400 μL of Assay Buffer was introduced into the reference sector, and 400 μL of sample was placed in the sample sector. Centrifugation continued for about 16 h, and concentration-profile data were collected using both absorbance and interference optics. Data were initially analyzed using the n SEDFIT ,83. As wn SEDFIT .For difference SV studies, a slightly different protocol was followed. First, a 5 mg/mL stock of TpMglB-2 (in Assay Buffer) was made, and stock solutions of the sugars to be examined were also prepared in Assay Buffer. Identical volumes of the sugars were dispensed to separate tubes, followed by the addition of identical volumes of the protein stock solution. The two pairs of sugars studied were D-mannose/D-glucose and D-ribose/D-glucose. In a third experiment (a negative control), no sugars were added, only Assay Buffer. The solution containing the non-binding sugar was placed into the reference sector of a centerpiece, and the solution containing D-glucose was inserted in the sample sector. For the D-mannose/D-glucose and buffer/buffer experiments (performed on different days), the experiment was carried out in a meniscus-matching centerpiece . Thus, ca. 10 μL less of the glucose containing solution was dispensed than that of the reference-sector solution. The solution-column heights were equalized prior to the experiment by 30 min of centrifugation at 9,000 rpm. After equalization, the centrifugation cell was removed and repeatedly inverted to thoroughly mix the sector contents. The equalization step resulted in a small amount of non-binding sugar being transferred to the sample sector. The D-ribose/D-glucose experiments were done in a normal Beckman-Coulter centerpiece, so the column-height equalization step was not possible. A deliberate meniscus mismatch was introduced into this experiment by pipetting ca. 6 μL less into the sample sector compared to the reference sector, as suggested . The samJ, on the y-axis and the radius from the center of rotation on x-axis (r). In this case, of course, the difference in fringe displacement, ΔJ, was measured. Difference SV data, as mentioned above, take on the appearance of a Gaussian distribution inverted about the x-axis  and J curve. These data were fitted to a straight line, and the slope was taken as the quantity As presented by the data-acquisition software, interference data have the amount of fringe displacement, e x-axis . The detelsewhere. Estimatchachman .www.biolog.com).The thermal stability of TpMglB-2 in the presence of various putative ligands was determined in a 96-well PCR-plate . The assumption was that binding ligands would enhance the thermal stability of the protein. For carbon-source screening, Biolog's Phenotype MicroArray (PM) compounds supplied in 96-well microplates  were dissolved in 50 μL of sterile water to obtain a final concentration of around 10–20 mM. Screenings were performed with plates PM1 and PM2A. Each plate contains 95 compounds and a blank (no ligand) control. The complete plate contents are available at the BIOLOG website . Significant positive shifts in Tm, app were taken as positive results and were confirmed using ITC.Each 20 μL standard assay mixture in a 96-well PCR-plate contained 10 μM purified protein and SYPRO Orange (Life Technologies) at 5x concentration in a buffer containing 10 mM phosphate, 100 mM NaCl, pH 7.4. Two μL of the resuspended BIOLOG compounds were added to each well. The plate was sealed and placed in a BioRad CFX96 real-time PCR detector coupled to a C1000 thermal cycler , and the fluorescence of the dye was monitored as a function of temperature from 4° C to 95° C. Fluorescence readings were recorded every 0.5° C. The data were transformed to–dF/dT v. T curves, and the abscissas of the minima in these curves were defined as the apparent melting temperatures (E. coli and S. typhimurium). PROMALS-3D [BLAST  was usedOMALS-3D  was usedOMALS-3D  was applOMALS-3D  version S1 FileFile S1_File.7z is a compressed folder containing the raw data for the AUC, ITC, and bioinformatics results described in this paper.(7Z)Click here for additional data file.


We analyzed the influence of prior exercise designed to reduce predominantly muscle glycogen in either type I or II fibers on pacing and performance during a 4-km cycling time trial (TT). After preliminary and familiarization trials, in a randomized, repeated-measures crossover design, ten amateur cyclists performed: 1) an exercise designed to reduce glycogen of type I muscle fibers, followed by a 4-km TT (EX-FIB I); 2) an exercise designed to reduce glycogen of type II muscle fibers, followed by a 4-km TT (EX-FIB II) and; 3) a 4-km TT, without the prior exercise (CONT). The muscle-glycogen-reducing exercise in both EX-FIB I and EX-FIB II was performed in the evening, ∼12 h before the 4-km TT. Performance time was increased and power output (PO) was reduced in EX-FIB I (432.8±8.3 s and 204.9±10.9 W) and EX-FIB II (428.7±6.7 s and 207.5±9.1 W) compared to CONT , without a difference between EX-FIB I and EX-FIB II (P>0.05). The PO was lower in EX-FIB I than in CONT at the beginning and middle of the trial (P<0.05). The mean aerobic contribution during EX-FIB I was also significantly lower than in CONT (P<0.05), but there was no difference between CONT and EX-FIB II or between EX-FIB I and EX-FIB II (P>0.05). The integrated electromyography was unchanged between conditions (P>0.05). Performance may have been impaired in EX-FIB I due a more conservative pacing at the beginning and middle, which was associated with a reduced aerobic contribution. In turn, the PO profile adopted in EX-FIB II was also reduced throughout the trial, but the impairment in performance may be attributed to a reduced glycolytic contribution (i.e. reduced lactate accumulation). To date, the effects of reduced endogenous carbohydrate (CHO) stores on performance during a time trial (TT) has received limited attention While it seems clear that pre-exercise muscle glycogen availability affects the pacing strategy, it is not known if fiber-selective glycogen depletion will have different effects on pacing strategy. Due to the high-intensity at the beginning of a middle-distance cycling TT An interesting and practical manner to test the effects of fiber-selective glycogen depletion on pacing strategy would be to use validated protocols to reduce selectively the muscle glycogen content of type I or II fibers Therefore, the aim of this study was to analyze the influence of prior selective muscle glycogen-lowering exercise, designed to predominantly reduce the muscle glycogen content of either type I or II fibers, on pacing strategy and performance during a 4-km cycling TT. We hypothesized that the exercise protocol designed to predominantly lower the muscle glycogen content of type I fibers would impair PO during the midpoint of the TT, while the exercise protocol designed to predominantly lower the muscle glycogen content of type II fibers would impair PO at the beginning and during the last part of the TT. In order to provide some insights about muscle recruitment This study was approved by the Ethics Committee of the Federal University of Alagoas.2peak) 4.1±0.1 L·min−1 (55.3±2.6 mL·kg−1·min−1), peak PO (PPO) 253.5±8.6 W] took part in this study. The athletes trained regularly (4–5 days per week) for more than 60 minutes per day and cycled more than 200 km per week. The sample size required was estimated, using mean power output (i.e. performance) as the main outcome, from the equation n = 8e2/d2, as suggested by Hopkins n, e, and d denote predicted sample size, coefficient of variation, and the magnitude of the treatment effect, respectively. Coefficient of variation was assumed to be 1.9% Ten amateur, competitive cyclists  were measured through enzymatic colorimetric reactions in a spectrophotometer .To obtain the EMG signal, the skin of the vastus lateralis muscle of the right leg was shaved, abraded, and cleansed with isopropyl alcohol. Then, bipolar surface silver-silver chloride electrodes , at a 20-mm interelectrode distance, were placed over the muscle belly and aligned to the muscle fiber direction. Before the TT, four 5-s submaximal isometric contractions of the knee  were performed as a warm-up aer and Pan, respectively) were estimated from calculations using the metabolic power and gross mechanical efficiency, in accordance with Hettinga et al. met was calculated from the following equation 2 is the oxygen uptake in L·min−1 and RER is the respiratory exchange ratio. During the warm-up for each experimental trial, the gross mechanical efficiency was determined using the following equation:warm-up is the PO in the warm-up . As gross mechanical efficiency was not different across the conditions, the values were averaged for subsequent analyses. The Paer during the TT was calculated by the equation:aer is the aerobic mechanical power output. An RER equal to 1.00 was assumed during the TT to calculate Pmetan was calculated from the following equation:an is the anaerobic mechanical power output, PO is the power output and Paer is the aerobic power output.The PO corresponding to aerobic and anaerobic contribution . To allow magnitude-based inferences about practical effects of prior low or high-intensity exercises on 4-km cycling time-trial performance, the uncertainty in the effects were expressed as likelihoods that the true value of the effects represent substantial changes  Data distribution was verified by Shapiro-Wilk’s test. Means of the performance time, PO, PThe maximal voluntary contraction carried out immediately before each TT was not significantly different between the three experimental conditions , indicating that there was no influence  from the exercise protocol performed in the evening before the TT.aer, Pan, and iEMG are shown in aer in the EX-FIB I was significantly lower than in the CONT (P<0.05), but there was no difference between CONT and EX-FIB II or between EX-FIB I and EX-FIB II (P>0.05). Mean Pan and iEMG were similar for all conditions (P>0.05).The means for performance time, PO, PThere were significant main effects for condition (P<0.05), distance (P<0.05), and interaction effects (P<0.05) for PO . In the aer and Pan every 200 m are shown in aer (P<0.05), but there was no significant interaction (P>0.05). In all conditions, the Paer increased exponentially and stabilized after the first 800 m (P<0.05), but was systematically lower during EX-FIB I than in the control (P<0.05).The results concerning the Pan only for distance (P<0.05), but no condition (P>0.05) or interaction effect (P>0.05). In general, the Pan in the first 600 m and last 200 m was higher than the other intervals for all conditions  and supra VO2peak exercise  is impaired if athletes start the trial after a muscle-glycogen-lowering exercise protocol A lower mean PO and a longer time to completion of the TT was observed in EX-FIB I and EX-FIB II compared with CONT. Qualitative inferences indicated that these changes were ‘very likely’ harmful and ‘likely’ when EX-FIB I and EX-FIB II were compared to CON, respectively, suggesting that the impairments in performance were meaningful. Using time-to-exhaustion tests, it has previously been reported that performance during high-intensity exercise  of the TT, when the PO increased again in the last part, i.e. a U-shaped curve. This pacing model has previously been shown in studies investigating pacing during a TT of similar distance and duration aer and Pan for EX-FIB II, but Paer was systematically reduced during EX-FIB I , suggesting that selective muscle glycogen depletion may not produce a reduction in the anaerobic metabolism of the same magnitude as when both types of fibers are depleted concomitantly The Post-TT [La] was significantly lower in EX-FIB II than in the CONT condition. An interaction effect indicates that the increase in [La] with exercise was less pronounced during EX-FIB II than during the CONT or EX-FIB I. A previous study has suggested that an exercise protocol designed to reduce glycogen from both type I and II muscle fibers reduces the anaerobic capacity by close to 20% during a single high-intensity (175 to 400 W) square-wave exercise The iEMG of the vastus lateralis did not parallel the changes in PO throughout the trial. Specifically, iEMG remained unaltered throughout the trial . We [33]Finally, we found that although the overall TT performance was impaired in both reduced muscle glycogen conditions, force production  prior to the TT was similar between the three conditions. These results highlight the task-specific nature of fatigue and indicate that the mechanism(s) that dictate task failure during a TT may not necessarily be the same as those influencing pre-exercise maximal voluntary contraction performances. In other words, the specific mechanism responsible for performance reduction may be exercise task dependent A possible limitation of the present study is that we did not use a gold-standard technique  to quantify the muscle glycogen content. However, the muscle biopsy technique is invasive and could have interfered directly with the athletes’ pacing and overall performance. Furthermore, the effectiveness of the selective muscle glycogen-lowering exercises used in this study is well established The results of the present study suggest that prior, prolonged, low-intensity exercise (designed to reduce glycogen of type I muscle fibers) was associated with impairment in PO at the beginning and middle of a 4-km TT, and this was accompanied by a reduction in the aerobic contribution. On the other hand, the prior, short, high-intensity exercise (designed to reduce glycogen of type II muscle fibers) impaired the PO and reduced the plasma [La] accumulation. Irrespective of the inclusion of either a prior prolonged, low-intensity exercise or short, high-intensity exercise, an impairment in overall performance of a middle-distance cycling TT was evident, but the pacing adopted, as well as energy system alterations, occurred in a specific way. To the best of our knowledge, this is the first study demonstrating that the distribution of energy expenditure during a middle-distance cycling TT is dependent of the intensity and duration of the exercise carried out in the evening before.


There has been much hype and controversy over the so-called ‘Noakes’ diet. This diet advocates a low-cardohydrate, high-fat and high-protein intake. As previously reported in the Journal, Noakes has expressed the view that this diet coupled with exercise could have a favourable impact on lipid levels and potentially avoid the need for drug therapy.2I have recently seen a patient who had been diagnosed with dyslipidaemia several years ago and was receiving rosuvastatin 5 mg daily. A lipogram taken in April 2012 (on therapy) showed a total cholesterol (TC) level of 5.1 mmol/l and low-density lipoprotein cholesterol (LDL-C) of 2.9 mmol/l, respectively. After two months on the Noakes diet, she had the lipogram repeated. This now showed a TC of 12.9 mmol/l and LDL-C of 9.6 mmol/l, respectively. This was in the absence of diabetes mellitus and hypothyroidism.While one case does not make a scientific study, it should nevertheless alert practitioners involved in treating patients with dyslipidaemia and attempting to lower cardiovascular risk, that subjects on the Noakes diet should be cautioned as to the potential risks and have their lipid profiles closely monitored.


Objective. To explore the effects of electroacupuncture (EA) at ST36 (EA-ST36) and at Ashi acupoints (EA-Ashi) on skeletal muscle repair. Methods. Seventy-five rabbits were randomly divided into five groups: normal, contusion, EA-Ashi, EA-ST36, and EA at Ashi acupoints and ST36 (EA-AS). EA  was applied after an acute gastrocnemius contusion. The morphology of myofibers and neuromuscular junctions (NMJs) and expressions of growth differentiation factor-8 (GDF-8), acetylcholinesterase (AChE), Neuregulin 1 (NGR1), and muscle-specific kinase (MuSK) were assessed 7, 14, and 28 days after contusion. Results. Compared with that in contusion group, there was an increase in the following respective parameters in treatment groups: the number and diameter of myofibers, the mean staining area, and continuities of NMJs. A comparison of EA-Ashi and EA-ST36 groups indicated that average myofiber diameter, mean staining area of NMJs, and expressions of AChE and NRG1 were higher in EA-Ashi group, whereas expression of GDF-8 decreased on day 7. However, increases in myofiber numbers, expressions of MuSK and AChE, as well as decreases in GDF-8 expression, and the discontinuities were observed in EA-ST36 group on the 28th day. Conclusion. Both EA-ST36 and EA-Ashi promoted myofiber regeneration and restoration of NMJs. EA-Ashi was more effective at earlier stages, whereas EA-ST36 played a more important role at later stages. A contusion is a common sports injury and involves approximately 12.1% of all skeletal muscles disorders . In receOur preliminary study confirmed that EA at both Ashi acupoints and Zusanli (ST36) acupoint (EA-AS) could promote the regeneration of muscle  and imprIn order to understand more details of the treatment and provide a laboratory basis for making better clinical decisions, we designed the present study to (1) determine whether EA-ST36 or EA-Ashi could improve skeletal muscle and neuromuscular junctions (NMJs) repair and (2) investigate the individual characteristics of EA-ST36 treatment and EA-Ashi treatment in the repair in a rabbit model with a GM contusion. To attain these goals, we evaluated the morphology of myofibers and NMJs. Furthermore, the mechanisms of the treatments were assessed by the following parameters: the expression of growth differentiation factor-8 (GDF-8) for muscle fiber regeneration, acetylcholinesterase (AChE) content, muscle-specific kinase (MuSK), and Neuregulin 1 (NGR1) for NMJ restoration.n = 15), contusion group (n = 15), EA-Ashi group (n = 15), EA-ST36 group (n = 15), and EA-AS group (n = 15). All animals had access to food and water ad libitum in temperature (23 ± 1°C) and humidity (50 ± 5%) controlled rooms with 12-hour light-dark cycles. The experimental procedures were approved by the Ethical Committee of the Academy of Medical Sciences and were conducted in accordance with internationally accepted principles for laboratory animal use and care.Seventy-five New Zealand white rabbits  were randomly divided into five groups: the normal group  through the marginal vein of the ear. The hindlimb was set by extending the knee and dorsiflexing the ankle to 90° to fully display the GM. The crushing machine was used to create a contusion with the drop-mass technique using 9.555 J of energy. The injured area was approximately 1 cmTo keep the injury site dry and avoid infection, 25 g/L anerdian was applied topically once per day. The animals were treated with EA 24 hours after contusion. In the EA-ST36 group, the main needle , Zhongyan Taihe Medical Instruments Co. Ltd., Beijing, China) was inserted into ST36   of the nμm sections parallel to the direction of the myofibers. The sections were stored at 4°C after drying at room temperature for immunofluorescence staining. Another set of samples was fixed in 4% formalin for 3 days and then processed via gradient alcohol dehydration and paraffin embedding. Then cut into 5 μm sections, alcohol dewaxing and hydration were performed for hematoxylin-eosin (HE) and immunohistochemical staining. The remaining tissue portions were wrapped in foil, frozen in liquid nitrogen, and stored at −80°C for enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (QRT-PCR).Five rabbits from each group were sacrificed by the air embolism method randomly on the 7th, 14th, and 28th day after contusion. The injured area of the GM was cut vertically into four equal parts after being washed by prechilled PBS. The first tissue (approximately 1.5 × 1.0 × 0.5 cm) was fixed in 4% paraformaldehyde for 24 hours. Then, the specimen was placed in 15% or 30% sucrose solution (mass fraction) until it sank down to the bottom of the container. After being embedded by optimum cutting temperature compound , the samples were cut into 30 2O for 1 min. The sections were then dehydrated with two applications of 95% alcohol and two applications of 100% alcohol for 30 sec each. The alcohol was then extracted with two applications of xylene. One drop of mounting medium was added, and the samples were covered with a coverslip. The slides were recorded and analyzed by microscopy  with a 20x objective lens. Five random fields of each section were chosen for the analysis of morphological changes of injured skeletal muscles. The number of muscle fibers was recorded while the average diameter was measured in the obtained pictures using Image-Pro Plus Image .After hydrating the tissue, the slides were dipped into a Coplin jar containing Mayer's hematoxylin and agitate for 30 sec. The slides were stained with 1% eosin Y solution for 10–30 sec via agitation and then rinsed in H2O2 at room temperature for 10 min, rinsed with distilled water, and immersed in confining liquid for sealing. For the first antibody incubation, GDF-8 (1 : 50) rabbit polyclonal antibody  was incubated overnight at 4°C. After rinsing with distilled water, the second antibody, horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG , was incubated at 37°C for 30 min. After rinsing, histochemical stain 3,3′-diaminobenzidine  was applied for visualization at room temperature for 10 min. After recording, five fields of each section were chosen, and the mean optical density (MOD) was computed using IPP.Tissue sections were placed in 3% Hα-bungarotoxin (α-BTX) can bind to AChR. With help of fluorescent dye labeling, the morphological characteristics of NMJs can be observed and measured visually through fluorescent microscopy. The sections were washed with PBS fluid, and, then, marker profiles were obtained using Pap Pen, stained with α-BTX conjugated to rhodamine   and protected from light for 1 hour. The sections were mounted with fluorescence-free glycerol after being washed with PBS fluid. Digital images of the NMJs were obtained with an OLYMPUS FV1000 confocal laser-scanning microscope with an excitation of 480 nm (40x objective for viewing and 60x objective for representative images). Five random NMJs were captured for each section; the maximum intensity flat plane projection was made from Z-stacked images in Image J software (NIH). Only NMJs in a complete en face view were selected for analysis. A Gaussian Blur filter with σ = 2.00 was applied after background was subtracted and noise despeckled. Mean stained area was quantified from binary images . Boutons are located over stabilizing invaginations called junctional folds, where high-density clusters of acetylcholine receptors (AChRs) reside . α-bungaThe protein levels of AChE in the samples were quantified using ELISA. Homogenized tissues blending with equivalent 0.05 mol/L PBS (PH = 8) were coated to microtiter ELISA plates and incubated at 4°C overnight. After washing, the plates were blocked by a 1 h room temperature with 3% BSA. The AChE antibody  (0.2 mg/mL) was added to the plates, which were then incubated for 1 h at 37°C. HRP-conjugated anti-rabbit IgG was added to quantify the binding of the secondary antibody. After chromogenic assay by 0.03% o-phenylenediamine and stopping the reaction by the addition of 2 mol/L sulphuric acid, the optical density (OD) was measured on a microplate reader  at 492 nm. This experiment was carried out twice on different dates under uniform laboratory conditions to avoid internal variations and thus to determine the reproducibility of the assay.We used QRT-PCR to evaluate the RNA according to the fluorescence value of the amplified exponential phase. Samples were homogenized in Trizol reagent , and total RNA was extracted according to the manufacturer's instructions. Subsequently, RNA was reverse transcribed , and qRT-PCR was carried out with an ABI 7900HT Sequence Detection System  using SYBR green (SYBR FAST qPCR Kit Master Mix (2x) Universal, KAPA Biosystems, USA). Relative expression was determined by simultaneous comparison to the “housekeeping” gene, actin, using the geNorm software . Transcriptions for MuSK and NGR1 were assessed. The primer sets used for PCR amplification were as follows: Actin-F1, CACACTCCCGCTCAGCTCAC and Actin-R1 GCTTGCTCTGGGCCTCGT; NGR1-F, CTTCGCTGTGAGACCAGTTCAG and NGR1-R CCAGTGATGCTTTGTTGATGC; Musk-F TGTTCTCCTGCCTGAGCCTG and Musk-R TTGCGGGTAGGATTCCACTG. P < 0.05 was considered to be statistically significant.The results were presented as the mean ± standard deviation. The data from each time point were analyzed with the SPSS 20.0 statistical software . The normal distribution was analyzed using single factor analysis of variance. The groups were compared using the LSD method. The outcomes were evaluated using double-sided inspection.During muscle regeneration, new skeletal muscle fibers exhibited their central nuclei and then matured into periphery nucleated fibers. The regenerative myofiber cytoplasm and large hyperchromatic nuclei were identified in the center of cell using basophilic blue staining . The HE β super family and an important myostatin that not only inhibits the regeneration of myofibers but also stimulates the differentiation of myofibroblasts [GDF-8 was a muscle-specific factor in the TGF-roblasts . Positivα-BTX for quantification and visualization. NMJs contained many wrinkles and appear to be plump and large “rosette-shaped” under normal conditions. In the samples, the NMJs degenerated and fragmented into oval-shaped patches one week after contusion, resulting in many dispersed synapses. With AChR aggregation, some of the synapses connected to one another, similar to short rods, and became larger in size a week later. NMJs in the contusion area exhibited a higher number of folds and were better stacked on the 28th day . Reconstruction of NMJ function requires a large number of AChRs aggregated in the MEP. This process is mainly regulated by the Agrin/MuSK pathway . MuSK isClinically, EA is widely applied in skeletal muscle injury treatment , and resIn injured skeletal muscle, it is generally accepted that the Ashi acupoint is the injured region. To avoid new injury generated from needles inserted into injured area, Ashi acupoints were not located in the exact contusion midpoint, but rather 10 mm  from theAfter contusion, the number and diameter of muscle fibers in the treatment groups were significantly increased compared to the contusion group, indicating that both EA-ST36 and EA-Ashi could promote skeletal muscle regeneration. The diameter increased more significantly in the EA-Ashi group. This greater increase in diameter might be related to the fact that the electrodes were placed on the distal and proximal ends of the contusions, as muscle fibers might adaptively thicken against the mechanical stretching generated by electrodes , 30. In α-BTX was used to measure the MEP of the rabbit GM at different time points after muscle damage by confocal laser-scanning microscopy. The mean staining area and discontinuities of the MEPs were calculated. The discontinuities reflected the clusterization of AChRs, whereas the mean staining area represents the volume of AChRs in each NMJ. The majority of NMJs in damage muscle had degraded, appearing as scattered oval-shaped plaques at 7 days after injury, indicating secondary damage to the NMJs after muscle contusion. As AChRs gradually gathered, the NMJ was partially reconstructed after 2 weeks and some cracks appeared. The scattered AChRs connected into sheets, and the NMJ became oval at 4 weeks after contusion, although many NMJs exhibited shrinking and dispersed shapes. Meanwhile, the mean stained area of the NMJs had become larger and the discontinuities had decreased. Aside from shape changes, we also observed that AChE increased gradually from days 7 to 28, indicating that the function of injured NMJs had been restored step by step. In contrast, the NMJs in the treatment groups exhibited fuller shapes with more folds, larger mean staining area, lower discontinuities, and a higher content of AChE, indicating that EA may promote the survival of NMJs and thus play a pertinent role in accelerating their recovery. Previous studies have shown that EA could promote the survival of injured neurons after transection of the neural stem [The most important event in the restoration of a NMJ is the expression and aggregation of AChR on the MEP. In this study, stained ral stem  and thatral stem . These fFurthermore, increases in the mean stained area of the NMJs and AChE expression were observed in the EA-Ashi and EA-AS groups on days 7 and 14, compared to that of the other groups. The treatments in these groups may potentially be better at increasing the proliferation of AChR in the early stage of repair. Although the mean stained area in EA-ST36 group was lower, the continuities and AChE content increased on 14 and 28 days after contusion, suggesting that the clusterization of AChR and NMJ's function had been enhanced.We next explored the molecular mechanism of how this happens. As we know, AChR first proliferated after injury and then regressed following clusterization in the reconstruction and development processes of the NMJ . NRG1 isIn conclusion, in this study, we demonstrate via morphological and molecular biological approaches that both EA-Ashi and EA-ST36 have an effect on the regeneration of myofibers and restoration of NMJ, leading to muscle repair, possibly through the regulation of GDF-8, AChE, NRG1, and MuSK. EA-Ashi was more effective at earlier stages, whereas EA-ST36 was more effective later on. The combination of these two acupoints achieved a better effect than either one. Therefore, EA-AS is a suggested method for skeletal muscle injury treatment, which combines regional and distal acupoints. Additional molecular investigations are needed to clarify whether it is more effective to increase the stimuli of EA-Ashi and decrease EA-ST36 stimuli at earlier stages and reverse later on than the same stimuli of EA-AS.


P-element was horizontally transferred from a distantly related species into Drosophila melanogaster. Despite causing ‘hybrid dysgenesis’, a syndrome of abnormal phenotypes that include sterility, the P-element spread globally in the course of a few decades in D. melanogaster. Until recently, its sister species, including D. simulans, remained P-element free. Here, we find a hybrid dysgenesis-like phenotype in the offspring of crosses between D. simulans strains collected in different years; a survey of 181 strains shows that around 20% of strains induce hybrid dysgenesis. Using genomic and transcriptomic data, we show that this dysgenesis-inducing phenotype is associated with the invasion of the P-element. To characterize this invasion temporally and geographically, we survey 631 D. simulans strains collected on three continents and over 27 years for the presence of the P-element. We find that the D. simulans P-element invasion occurred rapidly and nearly simultaneously in the regions surveyed, with strains containing P-elements being rare in 2006 and common by 2014. Importantly, as evidenced by their resistance to the hybrid dysgenesis phenotype, strains collected from the latter phase of this invasion have adapted to suppress the worst effects of the P-element.In a classic example of the invasion of a species by a selfish genetic element, the  Drosophila melanogaster. We see that, as it spreads, the transposable element is associated with damaging effects, including sterility, but that the flies quickly adapt to the negative consequences of the transposable element.Some genes perform necessary organismal functions, others hijack the cellular machinery to replicate themselves, potentially harming the host in the process. These ‘selfish genes’ can spread through genomes and species; as a result, eukaryotic genomes are typically saddled with large amounts of parasitic DNA. Here, we chronicle the surprisingly rapid global spread of a selfish transposable element through a close relative of the genetic model,  Drosophila genes .P-element, a DNA transposon. P-element was acquired by D. melanogaster by horizontal transfer from a distant relative, D. willistoni . Differences between F1 females from reciprocal crosses, which have identical nuclear genotypes, are likely due to different maternal contributions to the egg cytoplasm and implicate a cyto-nuclear incompatibility, as in the D. melanogaster hybrid dysgenesis system . There is no relationship between dysgenesis and coverage for the reciprocal cross , or for coverage of other TEs in any cross . As a pP-element and Tom1; Tom1 is not significantly associated with cytotype , and there was no relationship between sequence coverage of Tom1 and the strength of dysgenesis, as described above . In contrast, the presence of P-elements is strongly associated with the DI cytotype, with products corresponding to P-element (a subset of which were verified by Sanger sequencing) amplified from all 35 DI strains. We were also able to amplify full-length P-element from 31 of these strains using standard PCR, and from the 4 remaining strains using RT-PCR—we were able to amplify all 4 exons of the P-element individually in all of the 35 strains. In contrast, no P-element could be amplified from any of the 98 DS strains . Using RT-PCR, we confirmed that full-length P-element is expressed in the 5 DI strains used in experimental crosses for cytotyping, and that no full-length transcript could be amplified from the DS strains used . In 18 of the 48 DR strains, we found only partial copies of the P-element, consistent with the production of piRNAs from degraded copies of the transposon [D. melanogaster, the maternal suppression of P-element sequence in a bi-directional piRNA cluster (one to which both sense and antisense piRNAs map) [Based on these three lines of evidence, we conclude that the troduced ,45. Constroduced , maternaansposon ,47. If, NAs map) –48, the D. melanogaster, the DS and DI strains might show temporal and/or geographical differences [z = 5.822, p = 5.81x 10−9), and previous work shows differences in P-element content between a samples from 2010 and 2012 [P-element .The fraction of strains containing the dominate . After 2elements . D. sechybridize ,54, remaP-elements in some form  grew about 4% per year from 1961–2013 (the years tracked by the Food and Agriculture Organization of the United Nations Statistics Division). As a result, the average tonnage of fruit imports was 5-fold higher in the years measured during the spread of the P-element in D. simulans compared to D. melanogaster . While these statistics capture only the movement of fruit between countries, the increase in movement seems likely to have boosted the global spread of the P-element in later years.Another, simpler explanation for the difference in the speed of invasion is higher migration rates between ear loci –61. FurtP-element in D. simulans adds to a handful of documented cases of the spread of a horizontally transmitted element through a Drosophila species [P-element appears to be spreading mainly via a selfish mechanism; though the occasional insertion may be beneficial [D. simulans and D. melanogaster have quickly evolved to suppress the dysgenesis phenotype by the end of the invasion , sugD. simulans isofemale lines collected across multiple locations over 15 years .We used Wolbachia infections before crosses were performed, in order to obtain sufficient offspring from crosses that normally show high levels of cytoplasmic incompatibility.  and wsp691R (5′-AAAAATTAAACGCTACTCCA-3′), and a cycling program of 94°C for 5' followed by 30 cycles of 94°C 30", 55°C for 60", 72°C for 2', and ending with one cycle of 72°C for 10', as in [Unless otherwise stated, all lines were cured of D. melanogaster [To assay for hybrid dysgenesis, we performed reciprocal crosses between lines using 5 virgin males and females from each line. Flies were left to lay eggs for a total of 9 days at 29°C, transferred to a fresh vial every 3 days, and offspring reared at 29°C . We To classify strains as DI, DR, or DS, we reciprocally crossed each strain to at least one known DI and DS ‘tester’ strains, and roughly classified tested strains based on the results of the four crosses. Specifically, we defined strain as:DI, if strain x tester DI, non-dysgenic; strain x tester DS, dysgenicDS, if strain x tester DI, dysgenic; strain x tester DS, non-dysgenicDR, if strain x tester DI, non-dysgenic; strain x tester DS, non-dysgenic.Some strains could not be unambiguously classified . These strains were not used in the analysis; in all, 181 strains were unambiguously cytotyped.D. melanogaster recovers with age [To perform the sterility assays shown in with age , we checD. simulans reference genome produced from a Madagascar strain [Drosophila TE sequences . Pairedr strain  and 179 se.org/; ,72) usinse.org/; ; parametD. simulans M252 reference genome using BWA-SW with the default parameters [D. simulans genome sequence, some insertion sites may not be recovered by PoPoolationTE, which relies on mapping reads to a reference. We therefore also estimated TE copy number per family by comparing average coverage of the TE sequence to average coverage of chromosome arm 2L.We examined whole genome sequence from the Florida strains using paired-end Illumina reads . The-element .P-elements was assessed using the forward primer for exon 0 and the reverse primer for exon 3, and using a primer annealing to both ends of the P-element . To ensure that PCR failures are due to the absence of P-element, DNA quality was checked with a positive control PCR for an essential gene, and D. melanogaster Harwich P was used as a positive control in all PCRs. Sanger sequencing  of a subset of PCR products was used to confirm that the amplicons correspond to P-element sequence (Genbank accession numbers KU719478-KU719502); the sequence of additional sequencing primers is available upon request. We required a minimum of two independent DNA extractions to consistent results for each line.The presence of full P-element RNA (using the forward primer for exon 0 and the reverse primer for exon 3): 45°C for 30' for reverse transcription, then 95C for 15', followed by 30 cycles of 94C for 15", 55C for 60" and 68C for 6'; 68C for 10' to amplify cDNA. Using the same program, reverse transcription was also performed separately for both forward and reverse of the P-element (using either the forward primer for exon 0 or reverse for exon 3) to generate unidirectional cDNA for PCR.To perform RT-PCR, RNA was isolated from Florida, Georgia, Harvard, Maryland and Madagascar lines using Peqlab TriFast RNA isolation protocol, and extractions checked on a 1% agarose gel following treatment with DNase. We then performed RT-PCR using QIAGEN OneStep RT-PCR Kit, using the following program to detect full-length https://www.r-project.org/). To estimate global trade in fruits, we downloaded data for total worldwide imports and exports of fruits and vegetables for the years 1961 to 2013 (measured in tons) from the FAOstat website (http://faostat3.fao.org/download/T/TP/E). From these, data for nuts, vegetables, and processed foods were excluded, resulting in data for 29 categories of fleshy fruit and berries. Using the nls function in R, we fit exponential growth models to these data of the form y = a exp(bx), where y is the exports in tons, x is the number of years since1961, and a and b are the estimated intercept and growth rate, respectively.All statistical analyses were performed using R ; https:/S1 Fig(A-F) and dysgenic ovaries (G-L). The brown bar on each image is a scale of 0.5mm. All ovaries were taken from F1 females from crosses between SGA35 & M252 for each direction. Non-dysgenic ovaries, though they appear to vary in size and ovariole number (e.g. C vs. D), are much larger than dysgenic ovaries. In dysgenic ovaries, if present, they are usually rudimentary and only one or two ovarioles form properly (I-L). Dysgenic ovaries can also be asymmetric, as ovarioles will occasionally form on one side but not the other (I-K), as previously described in D. melanogaster [Images showing non-dysgenic (TIF)Click here for additional data file.S2 Figp < 0.05). A) Initial crosses between Georgia (2009) and Madagascar (2004) flies at 29° C, with the fraction of dysgenic F1 females shown when the Georgia strain is paternal (positive direction) or maternal (negative direction). B) A subset of the crosses in panel A were repeated at 25°C, a non-dysgenic temperature in D. melanogaster; repeated crosses are shown in the same position as in panel A. C) Fraction of dysgenic offspring from crosses within and between Georgia and Florida lines at 29°C. The first strain named in the cross is the paternal strain in the positive direction and the maternal strain in the negative direction. D) Fraction of dysgenic offspring from crosses within and between Madagascar lines at 29°C. The first strain named in the cross is the paternal strain in the positive direction and the maternal strain in the negative direction. E) Fraction of dysgenic offspring from crosses between Georgia and Madagascar lines at 29C after curing of Wolbachia with tetracycline hydrachloride for 2 generations. F) Fraction of dysgenic offspring from crosses between Georgia and Madagascar lines at 29C before curing.Bar plots show the fraction of dysgenic offspring for both directions of each set of reciprocal crosses. Bars are coloured red when a significant difference between each reciprocal cross is found Click here for additional data file.S3 Fighttp://dx.doi.org/10.5061/dryad.1rq8f.Map shows the approximate location where strains were collected; pie charts show the proportion of each population which were dysgenesis-inducing (red), dysgenesis-resistant (orange) and dysgenesis-susceptible (yellow). The area of the pie chart is proportional to the number of strains sampled. For raw data see: (PDF)Click here for additional data file.S4 FigA-C show the number of sterile F1 offspring produced from the dysgenic (red) and reciprocal (grey) crosses , shown with 95% confidence interval. Crosses that show a significant difference in sterility between directions are marked with a star (Fisher’s Exact Test p < 0.05). Plots D-F show the number of offspring produced by the female offspring from both the dysgenic and reciprocal cross. Crosses that show a significant difference in the number of offspring for each direction are marked with a star (Wilcox Rank Sum Test p < 0.05). Female parents were dissected after crossing to confirm the presence of gonadal dysgenesis . A & D. Females were mated after 3–9 days of aging. B & E. Females were mated after 10–16 days of aging. C & F. Combined results for all crosses and ages.Plots (PDF)Click here for additional data file.S5 Figvs. coverage for TEs in M252 (2004). Here, coverage is defined as the average per base coverage of TE sequence, divided by the average per base coverage of chromosome 2R in the line. The Florida coverage is taken as the average coverage of 12 available lines . The Florida data  and Madagascar data (SRA:SRX504933) was mapped to the D. simulans reference genome alongside Flybase TE sequences to identify differences in coverage for these TEs. Only one TE was found in Florida but not in Madagascar, P-element.Average coverage for TEs in the sequenced Florida (2010) x Madagascar (2004) isofemale lines (PDF)Click here for additional data file.S6 Figvs. the fraction of dysgenic offspring observed in a cross (12 lines from Florida population [described in A) The correlation between the copy number of P-element and the proportion of dysgenic offspring from the paternal Florida crosses (p = 8.49e-11). The equivalent association between dysgenesis and P-element copy number for D. melanogaster is shown with clear points [B) The correlation between the copy number of P-element and the proportion of dysgenic offspring from the maternal Florida crosses (p = 0.259). C. The correlation between the copy number of Tom1 and the proportion of dysgenic offspring from the paternal Florida crosses (p = 0.581). D. The correlation between the copy number of Tom1 and the proportion of dysgenic offspring from the maternal Florida crosses (p = 0.893).Estimated copy number of TE (estimated via dividing the average coverage of the TE in a sample by the average coverage of chromosome 2R) r points . B) The (PDF)Click here for additional data file.S7 FigA) RT-PCR product from DI and DS lines. Group 1 consists of female flies from three DI lines roughly dissected into soma (S) and ovaries (G). The top band, at 2.6kb, corresponds to the expected size of the full-length P-element transcript after splicing. Group 2 consists of female flies from two DS lines with a positive control (D. melanogaster Harwich strain). B) PCR product from DI, DR & DS lines following reverse transcription with either forward (1) or reverse (2) primers.(PDF)Click here for additional data file.S1 TableTable includes time and date of collection, and collector. The fraction of strains with P-element are also shown for each collection.(PDF)Click here for additional data file.S2 TableNumber of reads mapping shows the number of reads which mapped to the TE in the expression data (SRA:PRJEB7936). The Z-value and P-value are from a binomial generalised linear model used to analyze the association between the TE copy number  and the number of hybrid dysgenesis seen in a reciprocal crosses to an M line see also ; TEs cau(PDF)Click here for additional data file.S3 TableThe model used fit the year and region from which strains were collect see , plus an(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.